Recent crystallographic studies have identified the surface residues on a TCR Va domain and a VbCb chain. Ongoing studies are likely to identify the contact residues involved in at least some TCR:ligand interactions. However, crystal structures can not define the thermodynamic contributions of specific contacts at the interface. This information, which can be obtained only through systematic mutational and binding analyses, is essential to understanding the energetics of T cell recognition. This proposal will use alanine scanning mutagenesis of a well-studied TCR system to analyze the role of specific TCR residues in binding to the conventional peptide/MHC ligand and to different superantigens. The results will be used to guide the discovery of high affinity TCR that might have utility as soluble antagonists of some T cell-mediated diseases. The project will examine the Vb8.2 Va3 TCR from the alloreactive CTL clone 2C. This receptor has the highest known affinity for a peptide/MHC ligand (KD = 0.1 mM for the QL9/Ld complex). The TCR of CTL 2C also binds to at least seven other ligands: the positively selecting class I product Kb and the Vb8.2 reactive superantigens SEB, SEC1, SEC2, SEC3, SPEA, and MAM. Thus, this TCR can be used to study a variety of TCR:ligand interactions. Toward this effort, we have shown that a single-chain VbVa TCR (scTCR) expressed in E. coli binds with the expected specificity to QL9/Ld. In preliminary studies, 13 single-site alanine mutants of this TCR have been produced and partially characterized. This approach will be continued with the production of about 50 additional mutants in all CDR and HV4 residues and several Vb FR residues. Binding properties of mutants will be analyzed by various assays, including surface plasmon resonance. Results will identify the: 1) epitopes recognized by four anti-TCR antibodies (anti-Vb8 MAbs: KJ16, F23.1, F23.2, and the clonotypic mAb lB2); 2) functional TCR contact residues with QL91Ld; and 3) functional TCR contact residues with SEB, SECl, SEC2, and SEC3. Finally, the 2C scTCR will be expressed in a yeast display system in an effort to isolate high affinity soluble TCR. The strategy will involve mutagenesis of regions at the TCR:ligand interface, followed by selection with fluorescently labeled QL9/Ld and SEC3 and cell sorting. Mutants will be characterized for binding affinities and inhibition of T cell recognition.